Vaccines made from Gram-negative bacteria have a well known tendency to cause endotoxic shock. This may result in abortion or death. Gram-negative bacteria release endotoxin from their outer membrane to a slight extent while they are alive, and dividing, but to a far greater extent during and after their death. Bacterial endotoxin is naturally present in tap water, where it is called pyrogen. To avoid the induction of fever, pyrogen-free water for injection is prepared by distillation or other methods of purification. In man the injection of as little as one endotoxin unit (EU) (approximate 0.1 nanogram, or 10.sup.-10 gram) may cause a transient rise in body temperature. In man and other animal, larger doses cause endotoxic shock and death.
The rabbit is similar to man in sensitivity to endotoxin, and the rabbit traditionally has been used to test human injectable products for pyrogenicity. Most other animal species are less sensitive. Horses and pigs are considerably more sensitive than most laboratory rodents. Thus, in the laboratory, only the rabbit is suitable for testing the endotoxic activity of veterinary injectables, although mice can be made relatively sensitive with drugs that alter macrophage function.
In endotoxin assays, the rabbit has largely been replaced by an in vitro test that is even more sensitive. This depends on the action of endotoxin on a fluid extracted from the horseshoe crab (Limulus amebocyte lysate, or LAL). The addition of endotoxin in trace amounts causes LAL to gel. Before the gel develops, the transparency of LAL changes in a way that can be measured by a spectrophotometer as increasing optical density.
The free-endotoxin content of cultures of Gram-negative bacteria, killed to make vaccines, varies according to the skill of the manufacturer. One ml. may contain as little as 20 micrograms (2.times.10.sup.-5 gram) or as much as one milligram (10.sup.-3 gram). Vaccine makers have used several methods to decrease free endotoxin. One involves harvesting the bacterial cells, by centrifuging or filtering, and discarding the endotoxin-rich culture fluid. Another involves adsorbing the cultures with insoluble aluminum (Al) or calcium compounds (carriers) such as aluminum hydroxide gel (Al gel).
Adsorbing with Al gel tends to remove most of the free-endotoxin from solution. Some manufacturers allow the gel, with the endotoxin and other culture products that are absorbed to it, to sediment before decanting the supernatant fluid to remove the remaining free-endotoxin. The bacteria may not be adsorbed to the gel, in which case they usually sediment, leaving the supernatant fluid clear. Decanting has to be delayed until everything has settled and the fluid is clear.
Whether the bacteria have been harvested from the culture, or sedimenting has taken place after adsorbing with an aluminum or calcium compound, the materials are then usually resuspended in a simple aqueous fluid (water, saline, or a buffer solution). Assays will then usually show a disappointing decrease in free-endotoxin content; the change is much less than calculated from the dilution factor on resuspension. This is because endotoxin continues to escape from the bacterial surface, and loosely bound endotoxin becomes desorbed from the carrier.
The two processes are sometimes combined; the bacteria are harvested from the culture medium, resuspended in aqueous fluid, and then adsorbed. It has been shown that adsorbing aqueous suspensions of harvested bacteria with conventional amounts of Al gel produces an undesirable result. For example, when an aqueous suspension of Salmonella choleraesuis was adsorbed with Al gel, 25% v/v, there was no detectable free-endotoxin but the ability of the preparation to immunize mice against S. choleraesuis was almost completely eliminated. As soon as the gel began to settle, the supernatant fluid was crystal clear, denoting total adsorption.
It was concluded from these observations that a simple aqueous fluid, virtually free of culture medium, would not use up any of the binding capacity of the gel. This would leave the gel in a highly avid state so that anything bindable would be very tightly bound. Thus, all endotoxin was removed from solution but apparently the bacteria were too tightly bound to be released after injection. This evidently interfered with immunization. The observation was repeated with an aqueous suspension of killed Pasteurella multocida cells containing toxoid. After adsorption with Al gel, 25% v/v, there was no detectable free-endotoxin but the preparation had a drastically diminished power to induce neutralizing antitoxin in guinea pigs.
The above demonstrates the two extremes of a range of conditions. At one extreme, where Al gel is added to a whole culture, the peptones, and other proteinaceous solutes in the culture fluid, saturate the binding sites on the gel so that a lot of material, including free-endotoxin, is bound loosely or not at all. At the other extreme, where Al gel is added to an aqueous suspension of bacteria, there are almost no proteinaceous solutes to react with the gel and it remains fully avid, tightly binding everything with an affinity for the gel, particularly endotoxin and bacterial cells. In this condition, the tightly bound bacteria and their antigenic products are not free to interact with the cells of a vaccinated animal's immunity system, and immunization is poor.
Thus, there is a need for a method that produces a condition between the extremes of the observed range, where the binding power of the Al gel would be moderate and the adsorption optimal. Most of the endotoxin would be firmly bound, making the vaccine safe, but the binding of bacterial cells and antigens would be loose enough to allow good immunization. This optimal condition would be achieved by suspending the bacteria in a dilution of the culture medium that would appropriately modulate the avidity or affinity of the Al gel. This gave rise to the term affinity-modulated adsorption process, or AMAP.RTM..
Experiments that are the hallmark of AMAP.RTM. in its original form were conducted. While keeping the concentration of Al gel constant, in this case 25% v/v, the dilution of culture medium was tinted to achieve optimal adsorption. The end-point of the titration was indicated by a free-endotoxin concentration between 20 and 500 EUs per ml., as assayed by the LAL method.
Experiments with a number of Gram-negative bacteria, including Salmonella choleraesuis, Bordetella bronchiseptica, and Pasteurella multocida, showed that, in the presence of Al gel 25% v/v, the end-point was usually achieved when the culture medium was diluted to give a total concentration of peptones and other proteinaceous materials of about 1% w/v. This usually required diluting the culture medium by a factor of 2.5 to 3.5. Vaccination of animals with the AMAP.RTM.-treated materials confirmed that there was no loss of antigenic potency, and no clinical evidence of reactions to endotoxin. The AMAP.RTM. optimum had been found.
The SmithKline Beecham Animal Health vaccine sold under the tradename Atrobac 3 (bordetella, pasteurella and erysipelothrix), sold for the prevention of atrophic rhinitis and erysipelas in swine, is the first commercial product made by AMAP.RTM.. The process is applied to the two Gram-negative components, bordetella and pasteurella. The product has achieved a good reputation for efficacy and freedom from systemic reactivity (endotoxic shock).
There are other methods of controlling free-endotoxin in Gram-negative bacteria. One consists of a mild alkaline hydrolysis. For example, a culture may be heated at 80.degree. C. and a pH of 10. This treatment inactivates the endotoxin but also destroys many bacterial antigens, especially the proteins.
Another method is fairly effective but has limited application. It consists of the use of glutaraldehyde to inactivate the culture. Glutaraldehyde is a potent cross-linking agent and it binds most of the endotoxin in the culture. After inactivation with glutaraldehyde, bordetella cultures have a free-endotoxin content of roughly 1 microgram (10.sup.-6 gram) per ml. Glutaraldehyde, however, can be used only in cultures in synthetic growth media. In natural media the proteinaceous solutes bind the glutaraldehyde and prevent its action on the bacteria. The use of glutaraldehyde to make safe bordetella vaccines is described in U.S. Pat. No. 4,888,169, issued Dec. 19, 1989. "Bordetella bronchiseptica vaccine.")
The original version of AMAP.RTM. (AMAP.RTM., Mark 1) is characterized by the titration of culture medium against a conventional amount of Al gel to optimally modulate the avidity of the gel. AMAP.RTM., Mark 1, fulfilled the objective of eliminating endotoxic shock without decreasing antigenic potency.
Investigators in this area have recently become acutely aware of a serious problem with all bacterial vaccines containing conventional amounts of Al gel (roughly 10 to 25% v/v), regardless of AMAP.RTM. but including products made by AMAP.RTM.. These vaccines produce what is called a depot effect. The Al gel, or other mineral carrier, is not readily metabolized, and so it tends to remain in the tissues at the injection site. The bacterial cells and metabolic products adsorbed to the gel are then trapped in the tissues. There they induce chronic irritation leading to granulomas, abscesses, and ultimately scarring. This is especially serious when the vaccine is injected into the muscle of an animal raised for meat. At slaughter the affected cut of meat is often condemned and lost. This is called trim loss due to carcass blemish. The emergence of the injection-site-reaction problem plainly indicated that a new version of AMAP.RTM. was needed that would not cause appreciable local reactivity.